Primitivo Caballero

A screening of five Bacillus thuringiensis Vip3A proteins for their activity against lepidopteran pests

Five Bacillus thuringiensis Vip3A proteins (Vip3Aa, Vip3Ab, Vip3Ad, Vip3Ae and Vip3Af) and their corresponding trypsin-activated toxins were tested for their toxicity against eight lepidopteran pests: Agrotis ipsilon, Helicoverpa armigera, Mamestra brassicae, Spodoptera exigua, Spodoptera frugiperda, Spodoptera littoralis, Ostrinia nubilalis and Lobesia botrana. Toxicity was first tested at a high dose at 7 and 10 days. No major differences were found when comparing protoxins vs. trypsin-activated toxins. The proteins that were active against most of the insect species were Vip3Aa, Vip3Ae and Vip3Af, followed by Vip3Ab. Vip3Ad was non-toxic to any of the species tested. Considering the results by insect species, A. ipsilon, S. frugiperda and S. littoralis were susceptible to Vip3Aa, Vip3Ab, Vip3Ae and Vip3Af; S. exigua was susceptible to Vip3Aa and Vip3Ae, and moderately susceptible to Vip3Ab; M. brassicae and L. botrana were susceptible to Vip3Aa, Vip3Ae and Vip3Af; H. armigera was moderately susceptible to Vip3Aa, Vip3Ae and Vip3Af, and O. nubilalis was tolerant to all Vip3 proteins tested, although it showed some susceptibility to Vip3Af. The results obtained will help to design new combinations of insecticidal protein genes in transgenic crops or in recombinant bacteria for the control of insect pests.

Analysis of a naturally-occurring deletion mutant of Spodoptera frugiperda multiple nucleopolyhedrovirus reveals sf58 as a new per os infectivity factor of lepidopteran-infecting baculoviruses.

The Nicaraguan population of Spodoptera frugiperda multiple nucleopolyhedrovirus, SfMNPV-NIC, is structured as a mixture of nine genotypes (A-I). Occlusion bodies (OBs) of SfMNPV-C, -D and -G pure genotypes are incapable of oral transmission; a phenotype which in SfMNPV-C and -D is due to the absence of pif1 and pif2 genes. The complete sequence of the SfMNPV-G genome was determined to identify possible factors involved in this phenotype. Deletions of 4860 bp (22,366-27,225) and 60 bp (119,759-119,818) were observed in SfMNPV-G genome compared with that of the predominant complete genotype SfMNPV-B (132,954 bp). However no genes homologous to previously described per os infectivity factors were located within the deleted sequences. Significant differences were detected in the nucleotide sequence in sf58 gene (unknown function) that produced changes in the amino acid sequence and the predicted secondary structure of the corresponding protein. This gene is conserved only in lepidopteran baculoviruses (alpha- and betabaculoviruses). To determine the role of sf58 in peroral infectivity a deletion mutant was constructed using bacmid technology. OBs of the deletion mutant (Sf58null) were not orally infectious for S. frugiperda larvae, whereas Sf58null rescue virus OBs recovered oral infectivity. Sf58null DNA and occlusion derived virions (ODVs) were as infective as SfMNPV bacmid DNA and ODVs in intrahemocelically infected larvae or cell culture, indicating that defects in ODV or OB morphogenesis were not involved in the loss of peroral infectivity. Addition of optical brightener or the presence of the orally infectious SfMNPV-B OBs in mixtures with SfMNPV-G OBs did not recover Sf58null OB infectivity. According to these results sf58 is a new per os infectivity factor present only in lepidopteran baculoviruses.

Vip3C, a Novel Class of Vegetative Insecticidal Proteins from Bacillus thuringiensis

ABSTRACT Three vip3 genes were identified in two Bacillus thuringiensis Spanish collections. Sequence analysis revealed a novel Vip3 protein class (Vip3C). Preliminary bioassays of larvae from 10 different lepidopteran species indicated that Vip3Ca3 caused more than 70% mortality in four species after 10 days at 4 μg/cm2.

Natural populations of Spodoptera exigua are infected by multiple viruses that are transmitted to their offspring

Sublethal infections by baculoviruses (Baculoviridae) are believed to be common in Lepidoptera, including Spodoptera exigua. In addition, novel RNA viruses of the family Iflaviridae have been recently identified in a laboratory population of S. exigua (S. exigua iflavirus-1: SeIV-1; S. exigua iflavirus-2: SeIV-2) that showed no overt signs of disease. We determined the prevalence of these viruses in wild populations and the prevalence of co-infection by the different viruses in shared hosts. Infection by S. exigua multiple nucleopolyhedrovirus (SeMNPV) and iflaviruses in S. exigua adults (N=130) from horticultural greenhouses in southern Spain was determined using qPCR and RT-PCR based techniques respectively. The offspring of these insects (N=200) was reared under laboratory conditions and analyzed to determine virus transmission. Overall, 54% of field-caught adults were infected by SeMNPV, 13.1% were infected by SeIV-1 and 7.7% were infected by SeIV-2. Multiple infections were also detected, with 8.4% of individuals harboring SeMNPV and one of the iflaviruses, whereas 2.3% of adults were infected by all three viruses. All the viruses were transmitted to offspring independently of whether the parental female harbored covert infections or not. Analysis of laboratory-reared insects in the adult stage revealed that SeIV-1 was significantly more prevalent than SeMNPV or SeIV-2, suggesting high transmissibility of SeIV-1. Mixed infection involving three viruses was identified in 6.5% of laboratory-reared offspring. We conclude that interspecific interactions between these viruses in co-infected individuals are to be likely frequent, both in the field, following applications of SeMNPV-based insecticides, or in laboratory colonies used for SeMNPV mass production.

Intra- and Intergenerational Persistence of an Insect Nucleopolyhedrovirus: Adverse Effects of Sublethal Disease on Host Development, Reproduction, and Susceptibility to Superinfection

ABSTRACT Sublethal infections by Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) are common in field populations of the beet armyworm (S. exigua, Hübner) in the Almerian horticultural region of Spain. Inoculation of second, third, and fourth instars with occlusion bodies (OBs) of an isolate (VT-SeAl1) associated with vertically transmitted infections resulted in 15 to 100% of sublethal infection in adult survivors, as determined by reverse transcription-PCR (RT-PCR) detection of viral DNA polymerase transcripts, and quantitative PCR (qPCR) targeted at the DNA polymerase gene. The prevalence of adult sublethal infection was positively related to the inoculum OB concentration consumed during the larval stage. Sublethal infections persisted in OB-treated insects for at least five generations. Viral transcripts were more frequently detected in adult insects than in third instars. qPCR analysis indicated a consistently higher prevalence of sublethal infection than RT-PCR. Sublethal infection was associated with significant reductions in pupal weight, adult emergence, fecundity, and fertility (egg hatch) and significant increases in larval development time and duration of the preoviposition period. Insects taken from a persistently infected experimental population were significantly more susceptible to the OB inoculum than control insects that originated from the same virus-free colony as the persistently infected insects. We conclude that OB treatment results in rapid establishment of sublethal infections that persist between generations and which incur costs in the development and reproductive capacity of the host insect.

Genomic diversity in European Spodoptera exigua multiple nucleopolyhedrovirus isolates

Key virus traits such as virulence and transmission strategies rely on genetic variation that results in functional changes in the interactions between hosts and viruses. Here, comparative genomic analyses of seven isolates of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) with differing phenotypes were employed to pinpoint candidate genes that may be involved in host-virus interactions. These isolates obtained after vertical or horizontal transmission of infection in insects differed in virulence. Apart from one genome containing a piggyBac transposon, all European SeMNPV isolates had a similar genome size and content. Complete genome analyses of single nucleotide polymorphisms and insertions/deletions identified mutations in 48 ORFs that could result in functional changes. Among these, 13 ORFs could be correlated with particular phenotypic characteristics of SeMNPV isolates. Mutations were found in all gene functional classes and most of the changes we highlighted could potentially be associated with differences in transmission. The regulation of DNA replication (helicase, lef-7) and transcription (lef-9, p47) might be important for the establishment of sublethal infection prior to and following vertical transmission. Virus-host cell interactions also appear instrumental in the modulation of viral transmission as significant mutations were detected in virion proteins involved in primary (AC150) or secondary infections (ME35) and in apoptosis inhibition (IAP2, AC134). Baculovirus populations naturally harbour high genomic variation located in genes involved at different levels of the complex interactions between virus and host during the course of an infection. The comparative analyses performed here suggest that the differences in baculovirus virulence and transmission phenotypes involve multiple molecular pathways.

Consequences of interspecific competition on the virulence and genetic composition of a nucleopolyhedrovirus in Spodoptera frugiperda larvae parasitized by Chelonus insularis

Nucleopolyhedroviruses ( Baculoviridae ) are virulent insect pathogens that generally show a high degree of host specificity and have recognized potential as biological insecticides. Whenever viruses are applied for pest control, a proportion of the infected insects will also be parasitized by hymenopteran or dipteran parasitoids and interspecific competition for host resources will occur; the severity of such competition is likely to be modulated to a large degree by the virulence of each type of parasite. We examined the impact of parasitism by the solitary egg-larval endoparasitoid Chelonus insularis (Hymenoptera: Braconidae) on the speed of kill of nucleopolyhedrovirus-infected Spodoptera frugiperda (Lepidoptera: Noctuidae) larvae and the pattern of host growth and virus production in infected and/or parasitized hosts. We also examined the effect of parasitism on the virulence, infectivity and genetic composition of serially passaged virus. Both parasitism and viral infection resulted in a marked reduction in host growth. When third instar larvae were dually parasitized and virus-infected, the growth rate was even more severely affected compared to parasitized larvae. There was a significant increase in virus production in larvae infected at later instars. Interspecific competition resulted in a substantial decrease in pathogen production in parasitized larvae infected at the fourth instar, but not in parasitized larvae infected at earlier instars. The serial passage experiment resulted in the appearance of four distinct genetic isolates of the virus detected by restriction endonuclease analysis. Of the three isolates that appeared in nonparasitized larvae, two showed increased virulence, expressed by mean time to death, and for one of these the infectivity, expressed as LC 50 , was reduced. One isolate that appeared in parasitized larvae (isolate D) had increased virulence and infectivity. Southern blot analysis indicated that virus isolate D was most likely generated by point mutation of a restriction site or by alterations such as duplications, deletions or by recombination of two or more genotypic variants present in the wild-type nucleopolyhedrovirus isolate. Our study provides clear evidence of interspecific competition within the host, since, depending on the timing of inoculation, adverse effects were observed upon both the parasitoid and the virus.

Simultaneous occurrence of covert infections with small RNA viruses in the lepidopteran Spodoptera exigua.

Viral covert infections in invertebrates have been traditionally attributed to sublethal infections that were not able to establish an acute infection. Recent studies are revealing that, although true for some viruses, other viruses may follow the strategy of establishing covert or persistent infections without producing the death of the host. Recently, and due to the revolution in the sequencing technologies, a large number of viruses causing covert infections in all type of hosts have been identified. The beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae) is a worldwide pest that causes significant losses to agricultural and ornamental plant industries. In a previous project we used NGS to obtain a comprehensive transcriptome of the larval stage, revealing the presence of an important number of unigenes belonging to novel RNA viruses, most of them from the order Picornavirales. In order to characterize S. exigua viral complex, in this work we have completed the genomic sequences of two picorna-like viruses, and compared them to a SeIV1, a member of Iflaviridae previously described by our group. We performed additional studies to determine virus morphology, horizontal transmission, tissue and life stage distribution and abundance in the hosts. We discuss the role of virus persistent infections on insect populations.

Selection of a nucleopolyhedrovirus isolate from Helicoverpa armigera as the basis for a biological insecticide

BACKGROUND The cotton bollworm, Helicoverpa armigera, is an insect that causes damage in a wide range of crops in Spain. Seven isolates of H. armigera single nucleopolyhedrovirus (HearSNPV) from the Iberian Peninsula were subjected to molecular and biological characterization and compared with a Chinese genotype (HearSNPV-G4). RESULTS The estimated sizes of the Iberian genomes varied between 116.2 and 132.4 kb, compared to 131.4 kb of the HearSNPV-G4 reference genome. Phylogenetic analysis based on the lef-8, lef-9 and polh genes revealed that the Iberian strains were more closely related to one another than to other HearSNPV isolates. Occlusion body (OB) concentration-mortality responses (LC50 values) did not differ significantly among Iberian isolates when tested against a Helicoverpa armigera colony from Oxford (UK). Despite being the fastest killing isolate, HearSNPV-SP1 was as productive as isolates with lower virulence, with an average yield of 3.1 × 10(9) OBs larva(-1) . OBs of HearSNPV-SP1 and HearSNPV-G4 were similarly pathogenic against a recently established colony from southern Spain, although HearSNPV-SP1 was faster killing than HearSNPV-G4 against a range of instars. CONCLUSION The insecticidal properties of HearSNPV-SP1 mean that this strain is likely to prove useful as the basis for a biological insecticide for control of Helicoverpa armigera in Spain.

Gender-Mediated Differences in Vertical Transmission of a Nucleopolyhedrovirus

With the development of sensitive molecular techniques for detection of low levels of asymptomatic pathogens, it becoming clear that vertical transmission is a common feature of some insect pathogenic viruses, and likely to be essential to virus survival when opportunities for horizontal transmission are unfavorable. Vertical transmission of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) is common in natural populations of S. exigua. To assess whether gender affected transgenerational virus transmission, four mating group treatments were performed using healthy and sublethally infected insects: i) healthy males (H♂)×healthy females (H♀); ii) infected males (I♂)×healthy females (H♀); iii) healthy males (H♂)×infected females (I♀) and iv) infected males (I♂)×infected females (I♀). Experimental adults and their offspring were analyzed by qPCR to determine the prevalence of infection. Both males and females were able to transmit the infection to the next generation, although female-mediated transmission resulted in a higher prevalence of infected offspring. Male-mediated venereal transmission was half as efficient as maternally-mediated transmission. Egg surface decontamination studies indicated that the main route of transmission is likely transovarial rather than transovum. Both male and female offspring were infected by their parents in similar proportions. Incorporating vertically-transmitted genotypes into virus-based insecticides could provide moderate levels of transgenerational pest control, thereby extending the periods between bioinsecticide applications.